PROJECT SUMMARY/ABSTRACT The principal goal of this project is to develop pH-sensitive fluorophores to determine tumor microenvironment pH in breast cancer models using Cerenkov imaging. Increased acidity in the tumor microenvironment (TME) plays a role in the invasion and metastasis of cancer cells and contributes to chemotherapeutic resistance. This metabolic hallmark has sparked interest in the exploration of extracellular tumor pH measurement and imaging in vivo. The decrease in tumor microenvironment pH is due to a deregulated metabolism predominantly arising from increased glycolytic flux in cancer cells. Cerenkov radiation emitted by ?-particles is a potential tool for pH imaging in vivo, since pH-sensitive fluorophores or chromophores can selectively attenuate its multispectral emission, providing a quantitative readout. Cerenkov radiation occurs when subatomic particles like electrons and positrons emitted from decaying nucleotides travel faster than the speed of light in a dielectric medium. The high speed at which these subatomic particles travel results in the production of multispectral photons along the particle?s trajectory. This multispectral emission is continuous, includes the visible spectrum and can be selectively attenuated by pH sensitive fluorophores or chromophores at the absorption maxima of the compounds. The selective signal attenuation, known as selective bandwidth quenching can be quantified and correlated with a pH value, providing a non-invasive technique for pH imaging in vivo. This proposal will test the hypothesis that selective absorption of the Cerenkov multispectral emission by pH sensitive fluorophores will provide accurate pH measurements of the TME through Cerenkov imaging. The hypothesis will be tested by the following Specific Aims: Specific Aim 1: Synthesis and characterization of Cerenkov-active, pH sensitive fluorophores: This will include conjugation and chelation of selected pH-sensitive fluorophores with the metal chelators DOTA or NOTA and radiometals 68Ga and 90Y, in addition to chemical and optical characterization. Specific Aim 2: In vitro evaluation of Cerenkov-active, pH sensitive fluorophores: A pH titration curve will be constructed to correlate average radiance values obtained through Cerenkov imaging to pH values. Cell uptake and toxicity studies will be performed. Specific Aim 3: In vivo evaluation of Cerenkov-active, pH sensitive fluorophores: Synthesized probes will be injected intravenously into mice bearing breast cancer tumors. PET imaging will be used to assess biodistribution and probe delivery into tumors. Cerenkov imaging will be used to measure TME pH. Pharmacological modulation of lactate transport using syrosingopine will be done to alter the tumor pH and assess sensitivity of the Cerenkov imaging technique.